Team:BostonU/Workflow

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For a detailed example of our Chimera Characterization Workflow, please check out the <a href="https://2014.igem.org/Team:BostonU/ChimeraExample">Chimera Example</a> page. Below, we present a brief outline of the major steps involved in each stage (Design, Build, Test) of the Chimera workflow, along with a few high level examples. We also define what we consider Phase I, II, and III to be for our workflow.
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<h2>Phase I - Build and test basic parts.</h2>
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          Key software tools: TASBE Tools, Eugene (optional), Raven (optional)
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         <th scope="col"><center><h3>General Chimera Workflow</center></h3></th>
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         <td scope="col" width="50%"><center><h3>General Chimera Workflow</center></h3></td>
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<th scope="col"><center><h3>Case Study: BU Priority Encoder</h3></center>
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        <td scope="col" class="tableborderleft" style="padding-left: 15px"><center><h3>Case Study: BU Priority Encoder</h3></center>
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        <th scope="col" colspan="2"><h2>Phase I - Build and test basic parts.</h2><br>
 
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         <th scope="col">This notebook details the process undertaken to replace the high copy pMB1 origin in our existing MoClo Level 1 and Level 2 destination vectors (named DVL1 and DVL2, respectively) with lower copy origins. Namely, the ColE1 (~50 plasmids/cell), p15A (~10 plasmids/cell), and pSC101 (~5 plasmids/cell) origins were selected to replace the high copy origin in DVL1 and DVL2.<br><br>All protocols used in this notebook are found in our <a href="https://2014.igem.org/Team:BostonU/Protocols">protocols</a> section.</th>
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• Add parts to <a href="https://2014.igem.org/Team:BostonU/MoClo">MoClo library</a>. The following parts were found to be necessary for our priority encoder:<br><br>
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<p class="tab">• 3 MoClo level 1 and 3 MoClo level 2 backbones, each with a different <a href="https://2014.igem.org/Team:BostonU/Backbones">origin of replication</a>:<br></p>
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• ColE1<br>
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• p15A<br>
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• pSC101<br><br>
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<p class="tab">• 4 MoClo level 0 <a href="https://2014.igem.org/Team:BostonU/FusionProteins">fusion proteins</a>:<br></p>
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• <i>tetR</i>_GFP<br>
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• <i>tetR</i>_YFP<br>
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• <i>araC</i>_YFP<br>
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• <i>araC</i>_GFP<br>
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<br><br>
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<p class="tab">• X  MoClo level 0 <a href="https://2014.igem.org/Team:BostonU/ProjectTandemPromoters">tandem promoters</a>:<br>
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• pTet_pBad<br>
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• pBad_pTet<br>
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These parts were cloned into a <i>E. coli</i> Bioline strain using our MoClo and transformation protocols. They were purified and sequenced. Additionally, we built testing devices for each of the new parts. Details can be found on the <a href="https://2014.igem.org/Team:BostonU/FusionProteins">fusion proteins </a>, <a href="https://2014.igem.org/Team:BostonU/ProjectTandemPromoters">tandem promoters </a>, and <a href="https://2014.igem.org/Team:BostonU/Backbones">origin of replication</a> project pages. We tested these using our <a href="https://static.igem.org/mediawiki/2014/7/7c/Flow_Cytometer_WorkflowYABU.xls">FACS Workflow</a> and our BD LSRFortessa flow cytometer.The TASBE Tools were then employed to characterize their expression.
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<br><br>
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        <td scope="col" colspan="2"><h2>Phase II - Build and characterize TU behavior.</h2>
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          Key software tools: TASBE Tools, Eugene, Raven
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</td></tr>     
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<tr>
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        <td scope="col" width="50%"><center><h3>General Chimera Workflow</center></h3></td>
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        <td scope="col" class="tableborderleft" style="padding-left: 15px"><center><h3>Case Study: BU Priority Encoder</h3></center>
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<center><img src="https://static.igem.org/mediawiki/2014/f/fb/Phase_II_Chimera_updated.png" width="100%"></center>
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</p> </td>
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• Run one-pot <a href="https://2014.igem.org/Team:BostonU/Multiplexing">Multiplexing MoClo reaction</a>. We initially multiplexed RBSs.
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<p class="tab">• Eugene was employed to visualize all possible part substitutions.<br>
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• Raven was employed to optimize the assembly of these combinations.</p><br><br>
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• Clone multiplexed reactions into Pro strain of <i>E. coli</i> using Pro Transformation protocol.<br><br>
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• Pick 20 colonies per plate, purify, and sequence.<br><br>
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• Test using flow cytometry workflow and analyze data using the TASBE Tools.<br><br>
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<br><br>
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        <td scope="col" colspan="2"><h2>Phase III - Test regulatory arcs and assemble final device.</h2>
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          Key software tools: TASBE Tools, Eugene, Raven
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</td></tr>     
 +
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<tr>
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        <td scope="col" width="50%"><center><h3>General Chimera Workflow</center></h3></td>
 +
 +
        <td scope="col" class="tableborderleft" style="padding-left: 15px"><center><h3>Case Study: BU Priority Encoder</h3></center>
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<tr>
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        <td scope="col">
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<center><img src="https://static.igem.org/mediawiki/2014/2/29/Phase_III_Chimera_updated.png" width="100%"></center>
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</td>
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<td scope="col" class="tableborderleft" style="padding-left: 15px">
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• Test individual TU regulatory arcs<br>
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• Use Eugene to plan final device topology.<br><br>
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• Use Raven to guide MoClo assembly of encoder.<br><br>
 +
• Clone multiplexed reactions into Pro strain of <i>E. coli</i> using Pro Transformation protocol.<br><br>
 +
• Pick colonies, purify, and sequence.<br><br>
 +
• Test using flow cytometry workflow and analyze data using the TASBE Tools.<br><br>
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      </tr>
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Latest revision as of 02:44, 18 October 2014



Workflow




For a detailed example of our Chimera Characterization Workflow, please check out the Chimera Example page. Below, we present a brief outline of the major steps involved in each stage (Design, Build, Test) of the Chimera workflow, along with a few high level examples. We also define what we consider Phase I, II, and III to be for our workflow.

Phase I - Build and test basic parts.

Key software tools: TASBE Tools, Eugene (optional), Raven (optional)

General Chimera Workflow

Case Study: BU Priority Encoder

 • Add parts to MoClo library. The following parts were found to be necessary for our priority encoder:

• 3 MoClo level 1 and 3 MoClo level 2 backbones, each with a different origin of replication:

• ColE1
• p15A
• pSC101

• 4 MoClo level 0 fusion proteins:

• tetR_GFP
• tetR_YFP
• araC_YFP
• araC_GFP


• X MoClo level 0 tandem promoters:

• pTet_pBad
• pBad_pTet

These parts were cloned into a E. coli Bioline strain using our MoClo and transformation protocols. They were purified and sequenced. Additionally, we built testing devices for each of the new parts. Details can be found on the fusion proteins , tandem promoters , and origin of replication project pages. We tested these using our FACS Workflow and our BD LSRFortessa flow cytometer.The TASBE Tools were then employed to characterize their expression.

Phase II - Build and characterize TU behavior.

Key software tools: TASBE Tools, Eugene, Raven

General Chimera Workflow

Case Study: BU Priority Encoder

 • Run one-pot Multiplexing MoClo reaction. We initially multiplexed RBSs.

• Eugene was employed to visualize all possible part substitutions.
• Raven was employed to optimize the assembly of these combinations.



• Clone multiplexed reactions into Pro strain of E. coli using Pro Transformation protocol.

• Pick 20 colonies per plate, purify, and sequence.

• Test using flow cytometry workflow and analyze data using the TASBE Tools.

Phase III - Test regulatory arcs and assemble final device.

Key software tools: TASBE Tools, Eugene, Raven

General Chimera Workflow

Case Study: BU Priority Encoder

 • Test individual TU regulatory arcs
• Use Eugene to plan final device topology.

• Use Raven to guide MoClo assembly of encoder.

• Clone multiplexed reactions into Pro strain of E. coli using Pro Transformation protocol.

• Pick colonies, purify, and sequence.

• Test using flow cytometry workflow and analyze data using the TASBE Tools.







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